Nitroxide free radicals are under active investigation for their potential use as metabolically responsive
contrast agents in electron paramagnetic resonance and nuclear magnetic resonance imaging. The metabolism in human red blood cells of
lipid-soluble nitroxides, doxyl
stearic acids (DSA), has been investigated. We observed that under normoxia DSA were stable in red blood cells for at least 2 h, but
hypoxia stimulated
spin label reduction. Complete signal recovery after air or
ferricyanide oxidation suggested the formation of
hydroxylamine during
hypoxia. DSA reduction was found to be dependent upon the position of the
nitroxide ring in the
fatty acid chain with the reduction rate higher when the -NO degree of the doxyl ring was closer to the
fatty acid carboxylic end. The reduction kinetics of DSA with the doxyl ring nearest to the carboxylic end (5DSA) was bifasic. A rapid reduction of about half of the 5DSA was observed in the first hour and, thereafter, a slow reduction process become predominant. The slope of the slow reduction abruptly decreased below 5 microM, thus suggesting a concentration-dependent membrane-cytoplasm translocation of 5DSA. The reducing activity of the red blood cell (RBC) was completely recovered in the cell lysate. Under
hypoxia, purified
hemoglobin and
myoglobin reduced 5DSA and a complete recovery of the signal was obtained after air reoxidation.
Globin did not reduce 5DSA, while
methemoglobin showed only a small reduction of 5DSA, thus suggesting that ferrous-
heme was involved in the hypoxic reduction of DSA. both DSA localization and the characteristics of intracellular
reductant (
hemoglobin) are responsible for the high stability of DSA in the RBC.